The photoactive yellow protein (PYP) is a small cytoplasmatic protein capable of performing a photocycle when illuminated with blue light (λ max=446 nm). PYP has already been intensively studied with respect to its structural and biophysical features by several research groups (Borgstahl et al, 1995; Ujj et al, 1998; Perman et al., 1998; Genick et al., 1998). These studies have shown that this photoreceptor is a very good model to investigate the mechanism of light perception in biological systems. The chromophore of PYP is p-hydroxycinnamic acid, bound to a cysteine via a thioester bond. Upon illumination, PYP undergoes a photocycle which involves a trans-to-cis isomerisation of the chromophore. Analogous photocycles, although with a different chromophore, have been detected in e.g. bacteriorhodopsin, halorhodopsin, and the sensory rhodopsins (SRI and SRII) from Halobacterium salinarum. Several intermediates of the photocycle have been characterized, both photochemically and structurally. The PYP was first isolated from Halorhodospira halophila (Meyer, 1985). The similarity between the visible absorption spectrum of PYP and the wavelength dependence of the negative phototactic response implies PYP to be the receptor responsible for this effect (Sprenger et al., 1993). The protein was also found in Rhodospirillum salexigens and Halochromatium salexigens (Koh et al, 1996). Pyp-homologous genes were found in Rhodobacter sphaeroides (Kort et al., 1996) and Rhodobacter capsulatus (www.integratedgenomics.com). In Rhodospirillum centenum, a PYP-phytochrome chimera has been isolated, which is presumably involved in the regulation of the enzyme chalcone synthase (Jiang et al., 1999).
The biosynthetic pathway of p-hydroxycinnamic acid has been intensively studied in plants (Hahlbrock and Scheel, 1989; Dixon and Pavia, 1995; Campbell et al., 1996), in which trans-cinnamic acid is synthesized from L-phenylalanine by the action of a phenylalanine ammonia lyase (PAL). In the presence of a P450 enzyme system, t-cinnamic acid can be converted to p-hydroxycinnamic acid. Subsequently in this so-called ‘phenylpropanoid pathway’, the p-hydroxycinnamic acid is linked to coenzyme A by a p-hydroxycinnamyl:CoA ligase (pCL). The product formed in this pathway serves as an intermediate in plants for the production of various secondary metabolites, such as lignin and isoflavonoids.
Not only has PAL activity been found in plants (Koukol et al., 1961), it was also detected in fungi (Bandoni et al., 1968), yeast (Ogata et al., 1967) and Streptomyces (Emes et al., 1970). The gene sequence of pal from various sources has been determined and published (Edwards et al., 1985; Cramer et al., 1989; Louis et al., 1989; Minami et al., 1989; Anson et al., 1987, Rasmussen and Oerum, 1991). Studies of PAL from plants and micro-organisms have indicated that, in addition to its ability to convert L-phenylalanine to cinnamic acid, it can also accept L-tyrosine as a substrate. In these reactions the p-hydroxycinnamic acid is directly formed from L-tyrosine, without the formation of trans-cinnamic acid and without the intervention of a P450 enzyme system. In this case the enzyme is referred to as a tyrosine ammonia lyase (TAL). However, all eukaryotic PAL/TAL enzymes prefer the use of L-phenylalanine rather than L-tyrosine as their substrate. The level of TAL activity is always lower than PAL activity, but the magnitude of this difference varies over a wide range. As pointed out by Rösler et al. (1997), PAL and TAL activities reside on the same polypeptide in monocotylic plants. Both activities have similar catalytic efficiencies, in spite of large differences in KM and turnover numbers. The enzyme from dicotyledonous plants, on the other hand, only uses L-phenylalanine efficiently. Related to this enzyme an application was filed by Dupont (WO 02/10407 A1), entitled: ‘Bioproduction of para-hydroxycinnamic acid’. In essence, the construction of a TAL enzyme by mutagenesis of the yeast Rhodotorula glutinis PAL/TAL enzyme and production of the enzyme in Escherichia coli is claimed. The ratio of TAL activity to PAL activity is described to be 1.7.
No information was known about the occurrence of this enzyme in eubacteria, until we recently cloned and expressed a tyrosine ammonia lyase from Rhodobacter capsulatus (Kyndt et al, 2002). As described below, we showed that the catalytic efficiency of the Rhodobacter TAL for L-tyrosine was approximately 150 times larger than for L-phenylalanine under physiological conditions. This is the first enzyme that was found to have a larger specificity for L-tyrosine as substrate than for L-phenylalanine. After DNA sequencing it was found that there are four basepair differences, resulting in two differences in the translated protein sequence (His522→Asp and Ala535 deletion), as compared to the gene found in the Rhodobacter capsulatus genome sequencing project (www.integratedgenomics.com). We attributed these differences to either strain differences or genome sequencing errors. In the genome sequencing project the sequence in question is annotated as being a PAL, based on sequence homology.
pCL-activity has been found in several plants (e.g., Gross and Zenk, 1966; Lindl et al., 1972; Knobloch and Hahlbrock, 1977; Ehlting et al., 1999; Obel and Scheller, 2000). The enzyme catalyses the activation of various hydroxylated and methoxylated cinnamic acid derivatives to the corresponding thiol esters in a two-step reaction. During the first step, the coumaric acid and ATP form a coumaroyl-adenylate intermediate with the simultaneous release of pyrophosphate. In the second step, the coumaroyl group is transferred to the sulfhydryl group of CoA, and AMP is released. Despite their low overall sequence identity, one highly conserved peptide motif is common to pCLs, luciferases, fatty acyl-CoA synthetases and acetyl-CoA synthetases. This conserved, putative AMP binding domain has been used as the most important criterion to group these proteins in one superfamily, that of the adenylate-forming enzymes (Fulda et al., 1994).
Bacterial genes homologous to this second enzyme (pCL) were found downstream of the pyp gene in Halorhodospira halophila and Rhodobacter sphaeroides (Kort et al., 1996; Kort et al., 1998). During the sequencing of the Rhodobacter capsulatus genome a pcl homologous ORF was found (www.integratedgenomics.com). None of the gene products of these bacterial pcl sequences have been characterised, so it is not yet established whether or not coenzyme A is also the thiol containing substrate for the bacterial pCLs. Kort et al. (1996) suggested that “the pcl homologous gene product could be involved in an activation of the chromophore by the formation of a thioester bond with Coenzyme A”. He also suggested that the biosynthesis of p-coumaric acid, which in plants is performed by PAL, may consist of three consecutive steps in prokaryotes. If so, it was speculated that an aromatic aminotransferase, a 2-keto-acid reductase and a dehydratase, respectively, would be involved.
The present inventors have made it possible to clone and express the two biosynthetic genes (tal and pcl) of the photosynthetic bacteria Rhodobacter capsulatus in Escherichia coli. Until now, the only possible way to produce recombinant holo-PYP was to chemically attach the chromophore to the recombinant apo-PYP, as described by Imamoto et al. (1995) and Genick et al. (1997). The latter method was shown to have a lower yield of holo-PYP and may lead to non-specific reactions as compared to the present invention. We also found that the chemical reconstitution method failed when attempting to produce recombinant holo-PYP from Rhodobacter capsulatus, whereas the present invention is able to produce the holo-protein in large amounts.
The PYP cannot be produced in large amounts in natural genera, nor does the heterologous expression of the pyp gene alone in Escherichia coli and Rhodobacter sphaeroides lead to the formation of holo-PYP (Kort et al., 1996).